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ONLINE MUTATION REPORT
Clusters of non-truncating mutations of P/Q type Ca2+
channel subunit Cav2.1 causing episodic ataxia 2
E Mantuano, L Veneziano, M Spadaro, P Giunti, S Guida, M G Leggio, L Verriello, N Wood,
C Jodice, M Frontali
...............................................................................................................................
J Med Genet 2004;41:e82 (http://www.jmedgenet.com/cgi/content/full/41/6/e82). doi: 10.1136/jmg.2003.015396
E
pisodic ataxia type 2 (EA2, MIM 108500) is one of three
allelic disorders due to mutations of the CACNA1A gene
coding for the Cav2.1 subunit of P/Q type voltage gated
Ca2+ channels. The other two allelic diseases are familial
hemiplegic migraine (FHM, MIM 141500) and spinocerebellar ataxia type 6 (SCA6, MIM 183086). EA2 is characterised
by a complex and highly variable phenotype, widely overlapping that of SCA6.1 2 Its main features are episodes of
vertigo or ataxia of variable duration and frequency, a
permanent cerebellar deficit of variable severity, sometimes
progressive, and a cerebellar atrophy typically starting from
the anterior portion of vermis. Recently dyskynesia,3 muscular weakness,4 and epilepsy5 have been described in
association with EA2. FHM, on the other hand, is characterised by migraine attacks preceded by symptoms such as
unilateral limb paresis or paralysis, paraesthesias, and
dysphasia. Interictal cerebellar signs are reported in about
50% of patients.6
The CACNA1A gene product is the pore forming subunit of
P/Q type Ca2+ channels, expressed in the brain and
particularly in cerebellar Purkinje and granule cells, as well
as in neuromuscular junctions.7 8 The protein is predicted to
have four domains or repeats, each formed by six transmembrane hydrophobic segments (fig 1). Segments S5 and S6 of
each domain line the pore region. The S5–S6 linkers are
highly conserved sequences folded to leave their extremes in
the extracellular space, and place within the pore a P
sequence which exerts a critical role for ion selectivity and
permeation of the Ca2+ channel.9
Point mutations in the CACNA1A gene are responsible for
EA2 and FHM, whereas small expansions of a CAG repeat at
the 39 end of the gene characterise SCA6. FHM patients carry
exclusively missense mutations.10–14 EA2, on the other hand,
is reported in 17 cases5 14–20 as due to mutations truncating or
severely disrupting (TR) the protein sequence, and in seven
cases3 4 16 19 21–23 to non-truncating/disrupting mutations
(NTR), either missense or small in-frame deletions. Both
types of mutations lead to a loss of function, either
complete3 5 or partial.4 24 An EA2 phenotype was also found
in association with an expanded 20 CAG allele of the
CACNA1A gene25 and with a point mutation of the auxiliary
b4 subunit of the same P/Q Ca2+ channel, coded by gene
CACNB4.26
The present work, by substantially expanding the number
of EA2 NTR mutations of Cav2.1 subunit, shows their
tendency to cluster in specific protein regions, and analyses
their clinical phenotype in comparison with the phenotype
associated with TR mutations.
Key points
N
N
N
N
Episodic ataxia type 2 (EA2) is mostly due to loss of
function mutations that truncate or severely disrupt the
pore forming (Cav2.1) subunit of P/Q type Ca2+
channels, coded by the CACNA1A gene. Gain of
function missense mutations of the same gene are
responsible for familial hemiplegic migraine. In a few
cases, EA2 is due to mutations that do not truncate or
disrupt the Cav2.1 subunit.
Screening for CACNA1A gene mutations was carried
out for 27 patients with either typical EA2 or cerebellar
ataxia of no known genetic type.
Five new Cav2.1 non-truncating/disrupting mutations
were detected. From almost doubling the number of
these mutations, it clearly emerges that they have a
preferential location in specific protein regions, namely
S5–S6 linkers and their borders. Their associated
clinical phenotype is comparable with that reported for
carriers of truncating/disrupting mutations, but data
suggest a possible difference in age at onset and
frequency of mental retardation.
The results show that EA2 mutations that do not
truncate or disrupt the Cav2.1 subunit are not as rare
as previously thought. Their associated phenotype
might be less severe than that of truncating/disrupting
mutations. They are clustered in highly conserved
protein regions which may be particularly vulnerable to
amino acid changes and are likely to have a critical
role in the channel gating activity.
METHODS
different Italian centres and from the National Hospital,
Queen Square, London. All fulfilled minimal diagnostic
criteria, including episodes of ataxia or vertigo of variable
duration, interictal nystagmus, and absence of mutations in
SCA1, 2, 3, 6, 7, 8, 10, 12, 14, 17, and DRPLA genes. The
response to acetazolamide was not included among the
criteria, since in some cases the burden of the disease was so
mild that the side effects of treatment would not compensate
its advantages. Nonetheless, most patients (14/19) were
treated with acetazolamide and have responded positively in
terms of frequency and severity of attacks. Of the 19 EA2
patients, eight were sporadic cases and 11 had, overall, 20
first degree relatives with a similar disorder. These, appropriately informed, consented to participate in the research.
Since progressive ataxia is one of the features of EA2,1 21 a
Subjects
The screening for CACNA1A mutations was performed overall
for 27 index patients and 31 affected relatives; 19 were index
patients with a clinical diagnosis of EA2, collected from
Abbreviations: EA2, episodic ataxia type 2; FHM, familial hemiplegic
migraine; SCA6, spinocerebellar ataxia type 6; (N)TR, (non-)truncating/
disrupting; SSCP, single strand conformation polymorphism
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further eight index patients were selected from our series of
61 cerebellar ataxia (CA) patients who did not carry SCA 1, 2,
3, 6, 7, 8, 10, 12, 14, 17, or DRPLA mutation. Selection was
based on having a predominantly vermian cerebellar atrophy
and/or a history of vertigo/ataxia episodes preceding the
onset of a permanent ataxia, or of fluctuations of their
cerebellar symptoms. Two of the eight CA patients were
sporadic cases, and six had 11 first degree relatives with
vertigo episodes and nystagmus or permanent ataxia, who
consented, after appropriate information, to participate in the
research.
Mutation screening
Genomic DNA was isolated from peripheral blood leucocytes
using standard procedures. All 49 exons of the CACNA1A gene
and their intron boundaries were amplified by PCR using 55
primer pairs; 54 of these were previously described: exons 1–
46 and 47 short form, by Ophoff et al14; exons 37A, 42, and 47
long form by Jodice et al,25 and exon 37B by Trettel et al.27 In
addition, a primer pair (31B For 59-AACACGCCTCCCCAA
CTG-39; 31B Rev 59-GGAGATGCGTTCACAGTTAATG-39) was
designed on the basis of NT_031915 contig (actually
NT_011295), containing the CACNA1A gene, to amplify an
alternatively spliced little exon, coding for only two amino
acids (NP) and possibly responsible for P or Q specificity of
the channel.28 The 14 CACNB4 gene exons and their
boundaries were amplified by PCR using primer pairs
previously described for exons 3, 6, 9, 12, 13,26 and newly
designed for the other exons (web appendix).
Screening for mutations was performed by single strand
conformation polymorphism (SSCP) analysis. Denatured
PCR products were electrophoresed on Gene Gel Excel 12.5/
24 by GenePhor Electrophoresis Unit (Pharmacia Biotech,
www. bio-itworld.com). Conformers were revealed by the
silver staining method PlusOne (Pharmacia Biotech). PCR
products of patients with atypical migrating bands were
sequenced by the MWG BIOTECH Sequencing Service
(www.mwg-biotech.com), and checked for cosegregation
with the disease, whenever affected relatives were available.
The presence of the mutations in the general population was
tested by SSCP analysis in 65 randomly selected normal
subjects.
RESULTS
Mutation analysis
Of the 27 index cases from the two samples (EA2 and CA),
none was carrying CACNB4 mutations and six were carriers of
CACNA1A mutations (table 1). Four mutations (1, 2, 3, 4)
Online mutation report
were found in the EA2 and two (5, 6) in the CA group. Family
1 in table 1 with mutation F1491S was previously reported3
and will not be further considered, except with regard to the
frequency of detected mutations among our patients. All
mutations were carried by more than one affected family
member, except for index case 8 (R2136C), an adopted
person with no traceable parents. All were affecting highly
conserved amino acids (fig 2) and none of them was present
in 130 random chromosomes. Three of the four missense
mutations (table 1) are predicted to induce a drastic change
in amino acid characteristics: they lead to exchanges between
arginine (R), a large positively charged amino acid, and
cysteine (C) or glycine (G), which are small uncharged
residues, the first of which possibly is involved in the S–S
bond. Only one mutation (V1494I) substituted a highly
conserved amino acid with one having a higher molecular
weight but similar characteristics. The remaining mutation
was an in-frame deletion of two amino acids (DMS1488/89)
as a result of a six nucleotide deletion.
Figure 1 shows the locations of the five mutations and of
seven previously reported EA2 NTR mutations. From almost
doubling the number of NTR mutations, it clearly emerges
that they are preferentially located in the S5–S6 linkers (9/
12), and particularly of domain I and III (8/12), where they
tend to cluster at the extremities and their borders. Except for
E1757K, they do not directly affect the P sequence (fig 2)
responsible for ion selectivity and permeation into the cell,29
nor other functionally known sequences, such as the EFhand motif in the extracellular portion of III S5–S6 (fig 2),
thought to cooperate with the P sequence in selectively
binding Ca2+ ions.30 They do not affect, either, the residues
involved in channel inactivation (not shown in fig 2) in the
S5–S6 repeat I31 or the S6 repeat III.32
Three mutations are located outside these preferential
regions. The first one (DY1594;A1593D) in IV-S1 is associated
with a very mild episodic phenotype without permanent
signs.16 The second one (R1662H), without description of the
associated phenotype, is located in IV-S4.23 The last one
(R2136C), carried by one of our EA2 patients with a typical
EA2 phenotype, was located in the COOH tail upstream of the
polyglutamine repeat.
Clinical features
The main clinical features of the 11 patients carrying the
mutations identified in the present study are shown in
table 2. Their clinical picture was compared with that
described for carriers of TR mutations in several studies
(table 3). No statistical comparison, however, was attempted,
Figure 1 Predicted structure of the Cav
2.1 subunit of Ca2+ channels type P/Q
and sites of non-truncating/disrupting
(NTR) mutations causing EA2.
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Online mutation report
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Figure 2 Sequence (in single letter code) of the four S5–S6 linkers of human Cav 2.1 and their alignment with the same segments of other species and
of other human Ca2+ channels. P-sequences with ion permeation and selectivity function29 40 are in bold type. EF-hand-motif in repeat III30 is in bold
italic type. The residues mutated in EA2 patients are in bold type, enlarged and underlined. Mice mutations tg and rkr are in bold type, enlarged and
underlined for the mouse sequence only. The sequences of domain III include also the initial seven residues of the S6 region. Sequences are from
Swissprot Database: Human (HUM) Cav 2.1 ID O00555, Mouse (MOU) Cav 2.1 ID P97445, Rabbit (RAB) Cav 2.1 ID P27884, Drosophila (DRO) Cav
2.1 ID P91645, Human (HUM) Cav 2.2 ID Q00975, Human Cav2.3 ID Q15878.
since data from different studies might be collected and
reported according to differing methods and criteria. The
comparison will, therefore, provide only a very rough
indication of the overall clinical picture.
Tables 2 and 3 show that the age at onset of our patients
was on average at 17 years (95% confidence interval from 12
to 22). This is very similar to the mean (16 years) of 11
previously reported NTR patients3 4 19 21 22 and higher than
that in the TR group (mean = 9). In addition, the data
confirmed that vertigo/ataxia episodes are not always present
in EA2 patients, and the rate of patients without episodes
was similar in the two groups. Three of our patients (5mother, 6-proband, and 6-father), all from families not
originally diagnosed as EA2, reported no episodes. Two of
them experienced, however, paroxistic exacerbations of an
otherwise slowly progressive ataxia. The type and frequency
Table 1 Cav 2.1 non-truncating (NTR) mutations found
in 27 index patients
Family
DNA
Protein
ID
n
Nucleotide* Change
Exon
Change
Domain
4
5
3
1
6
2
3
2
2
2
3
1
1041
4722
4739-44
4747
4755
6681
5
28
28
28
28
45
C256R
G1483R
DMS1488/9
F1491S
V1494I
R2136C
I S5–S6
III S5–S6
III S6
III S6
III S6
COOH-ter
TGC-CGC
GGG-AGG
DGTCCAT
TTC-TCC
GTC-ATC
CGC-TGC
*Nucleotide numbers are based on the human CACNA1A sequence
(accession number AC X99897). Family already reported by Guida et
al.3 ID, identification; n, number of patients.
of symptoms during the episodes are comparable in the two
groups of patients. Headache, including migraine, was a
fairly common feature in both groups. Migraine, according to
international diagnostic criteria,6 was present in four of our
patients, only occasionally coinciding with vertigo/ataxia
episodes. Accurate interviews with these patients allowed
us to exclude any aura in three patients, and the fourth
reported a visual aura (flickering lights) without limb
weakness or paralysis, paraesthesias, or dysphasia.
Acetazolamide treatment was tried consistently in only five
cases from the group with an original diagnosis of EA2. A
positive response to acetazolamide, in terms of decrease of
episode duration and frequency, was obtained whenever
tried. The treatment was slightly more efficient in our
patients than in carriers of TR mutations, but the sample
sizes were too small to provide a meaningful indication.
Mild to severe interictal ataxia was present in a high
percentage of patients in both groups. Nystagmus was
observed in all of our patients, as expected, being one of
the diagnostic criteria. It was most frequently horizontally
beating on lateral gaze, but vertical or rotatory nystagmus
was observed in 2/11 patients, and in 4/11 it was in all
directions of gaze. Cerebellar atrophy at MRI was more
frequent in our patients compared with the TR group. This
difference, however, might be due to the small sample sizes
and/or to the inclusion in our group of CA patients, obviously
affected by cerebellar atrophy.
The frequency of the extracerebellar signs (tables 2 and 3)
was similar in the two groups of patients, but the type was
different. Mental retardation and/or learning difficulty is the
most frequent sign among TR carriers, but only one of our
patients showed a cognitive deficit (proband 2), and none of
the NTR carriers who were previously reported with a
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Online mutation report
Table 2 Clinical features in 11 patients carrying EA2 non-truncating CACNA1A mutations
Clinical feature
Age (years)
Age at onset (years)
Symptoms
Episodic
Frequency
Duration
Ataxia
Dysarthria
Vertigo
Nausea/vomiting
Oscillopsia
Headache
Migraine
Aura
Interictal cerebellar
Ataxia
Dysarthria
Dysmetria
Adiadochokinesia
Nystagmus
V atrophy
H atrophy
Extracerebellar
Acetazolamide
response
Mutation
#2
proband
#3
proband
#3
mother
#4
proband
#4
son
#4
son
#5
proband
#5
son
#6
proband
#6
father
#6
brother
54
25
32
8
64
15
49
10
17
10
13
10
57
24
28
22
34
28
60
2
38
20
+
monthly
mins//hrs
+
+
+
+
2
2
+
2
+
weekly
hrs
+
+
+
2
2
2
2
2
+
monthly
mins/hrs
2
2
+
2
2
2
2
2
+
weekly
hrs
+
+
2
+
+
2
2
2
+
weekly
hrs
+
+
2
+
+
2
2
2
+
weekly
hrs
+
+
2
+
+
2
2
2
fluctuats
monthly
hrs/days
2
2
2
2
2
2
+
auditory
+
yearly
secs/mins
2
2
+
2
2
+
2
2
fluctuats
monthly
hrs/days
2
2
2
2
2
2
+
2
2
2
2
2
2
2
2
2
2
2
2
+
variable
mins
2
2
+
2
2
2
+
2
mild
2
2
2
+
+
+
+
+
mild
2
2
2
+
+
2
2
+
mild
2
2
2
+
+
2
2
NT
mild
mild
2
2
+
+
+
2
+
2
2
2
2
+
NT
NT
2
+
mild
2
2
2
+
NT
NT
2
+
severe
mild
mild
mild
+
+
2
+
NT
2
2
2
2
+
NT
NT
2
NT
severe
mild
severe
mild
+
+
+
+
NT
mild
2
mild
mild
+
NT
NT
2
NT
2
2
2
2
+
NT
NT
2
NT
R2136C
DMS 1488/9 DMS 1488/9 C256R
C256R
C256R
G1483R
G1483R
V1494I
V1494I
V1494I
Mins, minutes; hrs, hours; secs, seconds; NT, not tested; V, vermis; H, hemisphere; fluctuations, fluctuation of permanent ataxia.
detailed clinical phenotype3 4 19 21 22 showed a cognitive
deficit. Proband 2 presented with a presenile mild dementia
and cortical-subcortical brain atrophy. Since she underwent a
thyroidectomy in the past, the cognitive deficit could be
related to hypothyroidism resulting from loose compliance or
imperfect control of the replacement therapy.35 36 The other
two patients of the NTR group with extracerebellar signs had
a sensorineural hypoacusia and a labyrinth areflectivity in
one case (fam 5-proband), and a complex ataxo-spastic
disorder in the other (fam 6-proband). The deafness of the
former patient was of cochlear origin, suggesting the
coexistence of Ménière’s syndrome (MIM 156000)33 or
non-syndromic deafness with vestibular dysfunction34
(DFNA9, MIM 601369) which, however, could not account
for the patient’s cerebellar features. Fam 6-proband presented with an ataxo-spastic clinical picture with exacerbations of her feeling of imbalance. The proband had inherited
the ataxo-spatic disease from the mother, presenting with the
same picture but without exacerbations, and the EA2
mutation from the father, who showed mild EA2 signs, as
did the proband’s brother. The mild EA2, paternally inherited
phenotype in the proband was probably obscured by the
maternally inherited disorder, except for the fluctuations of
the cerebellar symptoms.
Table 3 Frequency of main clinical features in carriers of Cav2.1 truncating/disrupting (TR) mutations reported in the
literature, and comparison with those found in carriers of non-truncating/disrupting (NTR) mutations in the present work
Patients with Cav 2.1 mutations
TR
Features
Ref
NTR
15
Patients (number)
1
Families (number)
1
Age*
38
Age at onset (range)* 1(1)
Episodes
1/1
ataxia
1/1
NV
1/1
Vertigo
0/1
VA`
0/1
Headache
0/1
Dysarthria
1/1
Response to
1/1
acetazolamide
Interictal signs
Cerebellar ataxia
1/1
Nystagmus
1/1
Atrophy at MRI
0/1
Extracerebellar
0/1
Cognitive
0/1
16
17
Ref
5
Ref Ref
20
6
40
10(2–19)
16/20
13/20
9–10/20
7–8/20
4/20
6–7/20
8–9/20
?
15
1
36
10(8–15)
?
?
?
?
?
11/15
?
7/8
1
1
?
8(8)
1/1
1/1
0/1
0/1
1/1
1/1
0/1
NT
12/19
13/19
2/4
10/20
9/20
10/14
13/15
3/3
0/15
0/15
1/1
1/1
0/1
1/1
1/1
Ref
18
2
2
36
1.5(1.5)
2/2
2/2
1/2
2/2
2/2
2/2
1/2
1/2
1/2
2/2
?
0/2
0/2
Ref
19
3
3
31
3.5(2–5)
3/3
3/3
2/3
1/3
1/3
1/3
0/3
2/3
2/3
2/3
?
1/3
1/3
Ref
20
1
1
41
10
1/1
1/1
1/1
0/1
0/1
0/1
0/1
1/1
0/1
1/1
0/1
0/1
0/1
Total
%
Our study
%
43
15
38
9(1–19)
24/28
0.8
21/28
0.7
14–15/28 0.5
10–11/28 0.4
8/28
0.3
21–22/43 0.5
10–11/28 0.4
12/15
0.8
11
5
41¡3
17¡2(8–28)
8/11
5/11
4/11
5/11
3/11
5/11
5/11
5/5
0.7
0.5
0.4
0.5
0.3
0.5
0.5
1.0
27/41
33/42
5/10
12/43
11/43
8/11
11/11
6/6
3/11
1/11
0.7
1.0
1.0
0.3
.1
.7
0.8
0.5
0.3
0.3
Only studies with a detailed description of the clinical phenotype were taken into consideration. *Mean¡SE in years; the exact number of patients showing a
specific symptom could not always be reconstructed, this particularly referring to one patient and the frequency with and without him/her is reported; `VA, visual
anomalies include oscillopsia, blurred vision, dyplopia; NT, not tested; ?, data not reported; NV, nausea and vomiting.
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Online mutation report
DISCUSSION
A screening for CACNA1A and CACNB4 mutations was
performed in two groups of index patients, one with EA2
and the other with CA of unknown genetic type. Mutations
were found only in the CACNA1A gene: overall, six mutations,
all of NTR type, were found in 27 index patients (one
mutation of this group of patients was already reported by
Guida et al3). Such a low detection rate has already been
reported in EA2 studies14 and might be due either to genetic
heterogeneity, or to phenocopies, or to mutations in the still
unexplored portions of the CACNA1A gene, such as the
expression regulatory regions.
The main cerebellar features, both paroxystic and interictal, in 11 carriers of these NTR mutations were comparable
with those previously reported in EA2 patients with TR
mutations. The comparison, however, suggests that the
former might have an earlier onset and a more frequent
cognitive deficit than the latter. The differences are maintained also when the previously reported NTR
patients3 4 19 21 22 are considered. At present, however, these
are just indications of possible trends differentiating the two
phenotypes, which must be confirmed by more accurate and
systematic assessments in a much larger patient population.
The present and previous EA2 NTR mutations appear to be
located at preferential sites of Cav2.1 protein, since most of
them cluster at the S5–S6 linkers and their borders, outside
the sequences with a known specific function. When
compared with FHM missense mutations, which are widely
scattered along S4, S5, S6, and their linkers in all 4 domains,
this appears to be a quite distinct feature of EA2 mutations.
Only two FHM mutations are in S5–S6 linkers, and are in the
selectivity filter sequence: T666M in domain II, associated
with an ataxic phenotype,12 and V1457L in domain III, with
no details of the associated clinical picture.11 Furthermore, it
should be noted that two out of three NTR CACNA1A
mutations carried by ataxic mice, tottering (tg) and rocker
(rkr), have a location similar to that of human NTR
mutations, namely tg in S5–S6 linker of the II repeat37 and
rkr in S5–6 linker of the III repeat38 (fig 2).
Of the three EA2 mutations located outside the preferential
areas, one (R2136C, identified in the present study) is in the
COOH tail upstream of the polyglutamine repeat. It is
noteworthy that a nearby mutation was detected in a patient
with a cerebellar deficit (P Giunti, unpublished data),
possibly indicating the site of a further cluster. The R2136C
mutation does not affect the binding sites for calmodulin and
auxiliary b subunits present in this protein region.28 It might,
however, affect the state dependent mobility of the COOH
tail—that is, its ability to cooperate in channel activity by
conformational changes,39 particularly in inactivation gating.32 The change from arginine to cysteine, probably involved
in S-S bonds, could limit the conformational mobility of the
C-terminus of the protein.
Four of the previously reported NTR mutations have been
functionally tested, and all were predicted to induce a loss of
channel function. F1491S was shown by Guida et al3 to
completely abolish the ion flux into HEK 293 cells, and in the
study by Jen et al4 F1406C appeared to decrease the current
density, leaving a small influx of divalent ions in COS7 cells.
G293R was shown to shift the current voltage relationship
toward more positive potentials and enhance inactivation in
Xenopus oocytes, as did mutation DY1594;A1593D.24 Although
no functional data are yet available for the remaining
mutations, their position in specific protein regions strongly
supports their functional relevance. It appears highly unlikely
that functionally irrelevant mutations, associated with the
same phenotype, will be found in clusters that include
mutations known to alter the channel function.
5 of 6
Recently, Jiang et al41 proposed a new model, based on a
protein structure analysis, of how membrane voltage gates
the pore in K+ channels. According to the model, the
membrane depolarisation induces S3 and S4 segments to
move within the membrane, from a position almost
perpendicular to the pore and near the intracellular surface,
to a position parallel to the pore near the extracellular
surface. The displacement of S4 opens the channel by pulling
S5 and S6 away from the axis of the pore. Should this model
hold also for Ca2+ channels, considering that voltage gated
channels share a common structure, this would imply that
S5–S6 linkers might have a highly relevant role in transmitting the correct gating movement from S5 to S6. Mutations in
the S5–S6 regions, hence, could interfere with channel gating
activity.
In conclusion, the present results show that EA2 NTR
mutations are not as exceptional as previously thought. Their
associated phenotype is likely to differ from that of TR
mutations by delaying age at onset and preventing cognitive
deficit. Their large majority clusters in at least two main
protein regions, providing indirect evidence of their functional relevance and indicating that these mutational hot
spots deserve a deeper analysis of their role in the channel
activity.
.....................
Authors’ affiliations
E Mantuano, L Veneziano, S Guida, M Frontali, Institute of
Neurobiology and Molecular Medicine, CNR, Rome, Italy
M Spadaro, Department of Neurological Sciences, La Sapienza
University, Rome, Italy
M G Leggio, Department of Psychology, La Sapienza University and
IRCCS Fondazione Santa Lucia, Rome, Italy
C Jodice, Department of Biology, Tor Vergata University, Rome, Italy
L Verriello, Clinica Neurologica, DPMSC Udine University, Udine, Italy
P Giunti, N Wood, Institute of Neurology, University College, London,
UK
This study was supported by MIUR (Italian Ministry of University and
Scientific Research)-PS-FISR (Neurobiotecnologie di Base e Applicate),
MIUR-FIRB (RBNE01XMP4_008) to MF, and Regione Calabria (contract
n˚6378).
Conflicts of interest: none declared
Correspondence to: Dr M Frontali, INeMM-CNR, Via Fosso del
Cavaliere, 00044 Frascati Rome, Italy; [email protected]
REFERENCES
1 Frontali M. Spinocerebellar ataxia type 6: channelopathy or glutamine repeat
disorder? Brain Res Bull 2001;56:227–31.
2 Mantuano E, Veneziano L, Jodice C, Frontali M. Spinocerebellar ataxia type 6
and episodic ataxia type 2: differences and similarities between two allelic
disorders. Cytogenet Genome Res 2003;100:147–53.
3 Guida S, Trettel F, Pagnutti S, Mantuano E, Tottene A, Veneziano L, Fellin T,
Spadaro M, Stauderman KA, Williams ME, Volsen S, Ophoff RA, Frants RR,
Jodice C, Frontali M, Pietrobon D. Complete loss of P/Q calcium channel
activity caused by a CACNA1A missense mutation carried by episodic ataxia
type 2 patients. Am J Hum Genet 2001;68:759–64.
4 Jen J, Wan J, Graves M, Yu H, Mock AF, Coulin CJ, Kim G, Yue Q,
Papazian DM, Baloh RW. Loss-of-function EA2 mutations are associated with
impaired neuromuscular transmission. Neurology 2001;57:1843–8.
5 Jouvenceau A, Eunson LH, Spauschus A, Ramesh V, Zuberi SM, Kullmann DM,
Hanna MG. Human epilepsy associated with dysfunction of the brain P/Qtype calcium channel. Lancet 2001;358:801–7.
6 The International Classification of Headache Disorders. Cephalalgia
2004;24:28–9.
7 Volsen SG, Day NC, McCormack AL, Smith W, Craig PJ, Beattie R, Ince PG,
Shaw PJ, Ellis SB, Gillespie A, Harpold MM, Lodge D. The expression of
neuronal voltage-dependent calcium channels in human cerebellum. Mol
Brain Res 1995;34:271–82.
8 Westenbroek RE, Sakurai T, Elliott EM, Hell JW, Starr TV, Snutch TP,
Catterall WA. Immunochemical identification and subcellular distribution of
the alpha 1A subunits of brain calcium channels. J Neurosci
1995;15:6403–18.
9 Armstrong CM, Hille B. Voltage-gated ion channels and electrical excitability.
Neuron 1998;20:371–80.
10 Battistini S, Stenirri S, Piatti M, Gelfi C, Righetti PG, Rocchi MD, Giannini F,
Battistini N, Guazzi GC, Ferrari M, Carrera P. A new CACNA1A gene
www.jmedgenet.com
Downloaded from http://jmg.bmj.com/ on June 17, 2017 - Published by group.bmj.com
6 of 6
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
mutation in acetazolamide-responsive familial hemiplegic migraine and
ataxia. Neurology 1999;53:38–43.
Carrera P, Piatti M, Stenirri S, Grimaldi LME, Marchioni E, Curcio M,
Righetti PG, Ferrari M, Gelfi C. Genetic heterogeneity in Italian families with
familial hemiplegic migraine. Neurology 1999;53:26–32.
Ducros A, Denier C, Joutel A, Vahedi K, Michel A, Darcel F, Madigand M,
Guerouaou D, Tison F, Julien J, Hirsch E, Chedru F, Bisgard C, Lucotte G,
Despres P, Billard C, Barthez MA, Ponsot G, Bousser MG, Tournier-Lasserve E.
Recurrence of the T666M calcium channel CACNA1A gene mutation in
familial hemiplegic migraine with progressive cerebellar ataxia. Am J Hum
Genet 1999;64:89–98.
Ducros A, Denier C, Joutel A, Cecillon M, Lescoat C, Vahedi K, Darcel F,
Vicaut E, Bousser MG, Tournier-Lasserve E. The clinical spectrum of familial
hemiplegic migraine associated with mutations in a neuronal calcium channel.
N Engl J Med 2001;345:17–24.
Ophoff RA, Terwindt GM, Vergouwe MN, van Eijk R, Oefner PJ,
Hoffman SMG, Lamerdin JE, Mohrenweiser HW, Bulman DE, Ferrari M,
Haan J, Lindhout D, van Ommen GJB, Hofker MH, Ferrari MD, Frants RR.
Familial hemiplegic migraine and episodic ataxia type-2 are caused by
mutations in the Ca2+ channel gene CACNL1A4. Cell 1996;87:543–52.
Yue Q, Jen CJ, Thwe MM, Nelson SF, Baloh RW. De novo mutation in
CACNA1A caused acetazolamide-responsive episodic ataxia. Am J Med
Genet 1998;77:298–301.
Denier C, Ducros A, Vahedi K, Joutel A, Thierry P, Rotz A, Castelnovo G,
Deonna T, Gerard P, Devoize JL, Gayou A, Perrouty B, Soisson T, Autret A,
Warter JM, Vighetto A, Van Bogaert P, Alamowitch S, Roullet E, TournierLasserve E. High prevalence of CACNA1A truncations and broader clinical
spectrum in episodic ataxia type 2. Neurology 1999;52:816–21.
Jen JC, Yue Q, Nelson SF, Yu H, Litt M, Nutt J, Baloh RW. A novel nonsense
mutation in CACNA1A causes episodic ataxia and hemiplegia. Neurology
1999;53:34–7.
Scoggan KA, Chandra T, Nelson R, Hahn AF, Bulman DE. Identification of two
novel mutations in the CACNA1A gene responsible for episodic ataxia type 2.
J Med Genet 2001;38:249–53.
van den Maagdenberg AM, Kors EE, Brunt ER, Van Paesschen W, Pascual J,
Ravine D, Keeling S, Vanmolkot KR, Vermeulen FL, Terwindt GM, Haan J,
Frants RR, Ferrari MD. Episodic ataxia type 2. Three novel truncating
mutations and a novel missense mutation in the CACNA1A gene. J Neurol
2002;249:1515–19.
Matsuyama Z, Murase M, Shimizu H, Aoki Y, Hayashi M, Hozumi I,
Inuzuka T. A novel insertion mutation of acetazolamide-responsive episodic
ataxia in a Japanese family. J Neurol Sci 2003;210:91–3.
Yue Q, Jen CJ, Nelson SF, Baloh RW. Progressive ataxia due to a missense
mutation in a calcium-channel gene. Am J Hum Genet 1997;61:1078–87.
Denier C, Ducros A, Durr A, Eymard B, Chassande B, Tournier-Lasserve E.
Missense CACNA1A mutation causing episodic ataxia type 2. Arch Neurol
2001;58:292–5.
Friend KL, Crimmins D, Phan TG, Sue CM, Colley A, Fung VS, Morris JG,
Sutherland GR, Richards RI. Detection of a novel missense mutation and
second recurrent mutation in the CACNA1A gene in individuals with EA-2
and FHM. Hum Genet 1999;105:261–5.
Wappl E, Koschak A, Poteser M, Sinnegger MJ, Walter D, Eberhart A,
Groschner K, Glossmann H, Kraus RL, Grabner M, Striessnig J. Functional
consequences of P/Q-type Ca2+ channel Cav 2.1 missense mutations
associated with episodic ataxia type 2 and progressive ataxia. J Biol Chem
2002;277:6960–6.
Jodice C, Mantuano E, Veneziano L, Trettel F, Sabbadini G, Calandriello L,
Francia A, Spadaro M, Pierelli F, Salvi F, Ophoff RA, Frants RR, Frontali M.
www.jmedgenet.com
Online mutation report
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6) due to
CAG repeat expansion in the CACNA1A gene on chromosome 19p. Hum
Mol Genet 1997;6:1973–8.
Escayg A, De Waard M, Lee DD, Bichet D, Wolf P, Mayer T, Johnston J,
Baloh R, Sander T, Meisler MH. Coding and non coding variation of the
human calcium-channel b4 subunit gene CACNB4, in patients with idiopathic
generalised epilepsy and episodic ataxia. Am J Hum Genet 2000;66:1531–9.
Trettel F, Mantuano E, Calabresi V, Veneziano L, Olsen AS, Georgescu A,
Gordon L, Sabbadini G, Frontali M, Jodice C. A fine physical map of the
CACNA1A gene region on 19p13.1–p13.2 chromosome. Gene
2000;241:45–50.
Soong TW, DeMaria CD, Alvania RS, Zweifel LS, Liang MC, Mittman S,
Agnew WS, Yue DT. Systematic identification of splice variants in human P/
Q-type channel alpha1(2. 1) subunits: implications for current density and
Ca2+ dependent inactivation, J Neurosci 2002;22:10142–52.
Tang S, Mikala G, Bahinski A, Yatani A, Varadi G, Schwartz A. Molecular
localization of ion selectivity sites within the pore of human L-type cardiac
calcium channel. J Biol Chem 1993;268:13026–9.
Feng ZP, Hamid J, Doering C, Jarvis SE, Bosey GM, Bourinet E, Snutch TP,
Zamponi GW. Amino acid residues outside the pore region contribute to Ntype calcium channel permeation. J Biol Chem 2001;276:5726–30.
Zhang JF, Ellinor PT, Aldrich RW, Tsien RW. Molecular determinants of
voltage dependent inactivation in calcium channels. Nature
1994;372:97–100.
Hering S, Berjukov S, Sokolov S, Marksteiner R, Weiss RG, Kraus R, Timin EN.
Molecular determinants of inactivation in voltage-gated Ca2+ channels.
J Physiol 2000;528:237–49.
Fransen E, Verstreken M, Verhagen WI, Wuyts FL, Huygen PL, D’Haese P,
Robertson NG, Morton CC, McGuirt WT, Smith RJ, Declau F, Van de
Heyning PH, Van Camp G. High prevalence of symptoms of Meniere’s disease
in three families with a mutation in the COCH gene. Hum Mol Genet
1999;8:1425–9.
Robertson NG, Lu L, Heller S, Merchant SN, Eavey RD, McKenna M, Nadol
Jr JB, Miyamoto RT, Linthicum Jr FH, Neto JFL, Hudspeth AJ, Seidman CE,
Morton CC, Seidman JG. Mutation in a novel cochlear gene causes DFNA9, a
human nonsyndromic deafness with vestibular dysfunction. Nat Genet
1998;20:299–303.
Belandia B, Latasa MJ, Villa A, Pascual A. Thyroid hormone negatively
regulates the transcriptional activity of the beta-amyloid precursor protein
gene. J Biol Chem 1998;273:30366–71.
Latasa MJ, Belandia B, Pascual A. Thyroid hormones regulate beta-amyloid
gene splicing and protein secretion in neuroblastoma cells. Endocrinology
1998;139:2692–8.
Fletcher CF, Lutz CM, O’Sullivan TN, Shaughnessy Jr JD, Hawkes R,
Frankel WN, Copeland NG, Jenkins NA. Absence epilepsy in tottering mutant
mice is associated with calcium channel defects. Cell 1996;87:607–17.
Zwingman TA, Neumann PE, Noebels JL, Herrup K. Rocker is a new variant of
voltage dependent calcium channel gene CACNA1A. J Neurosci
2001;21:1169–78.
Kobrinski E, Schwartz E, Abernathy DR, Soldatov NM. Voltage-gated mobility
of the Ca2+ channel cytoplasmic tails and its regulatory role. J Biol Chem
2003;278:5021–8.
Yang J, Ellinor PT, Sather WA, Zhang JF, Tsien RW. Molecular determinants
of Ca2+ selectivity and ion permeation in L-type Ca2+ channels. Nature
1993;366:158–61.
Jiang Y, Ruta V, Chen J, Lee A, MacKinnon R. X-ray structure of a voltage
dependant K+ channel. Nature 2003;423:33–41.
Downloaded from http://jmg.bmj.com/ on June 17, 2017 - Published by group.bmj.com
Clusters of non-truncating mutations of P/Q
type Ca 2+ channel subunit Cav2.1 causing
episodic ataxia 2
E Mantuano, L Veneziano, M Spadaro, P Giunti, S Guida, M G Leggio, L
Verriello, N Wood, C Jodice and M Frontali
J Med Genet 2004 41: e82
doi: 10.1136/jmg.2003.015396
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